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• W2079405562 abstract "Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1Nasmyth K. Haering C.H. The structure and function of SMC and kleisin complexes.Annu. Rev. Biochem. 2005; 74: 595-648Crossref PubMed Scopus (498) Google Scholar]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring. Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs [1Nasmyth K. Haering C.H. The structure and function of SMC and kleisin complexes.Annu. Rev. Biochem. 2005; 74: 595-648Crossref PubMed Scopus (498) Google Scholar]. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring. Cohesin is sumoylated between ATP binding and its hydrolysis during S phase Fusion of Scc1 to a Ulp domain effectively downregulates cohesin sumoylation Cohesin sumoylation is required for sister chromatid cohesion (SCC) and viability Sumoylation and Eco1-dependent acetylation act in parallel for SCC SUMO is conjugated to lysine residues by the sequential action of E1 and E2 enzymes (reviewed in [2Geiss-Friedlander R. Melchior F. Concepts in sumoylation: a decade on.Nat. Rev. Mol. Cell Biol. 2007; 8: 947-956Crossref PubMed Scopus (1353) Google Scholar]). Sumoylation is completely reversible, and deconjugation of SUMO is mediated by members of the Ulp/SENP family of peptidases [3Mukhopadhyay D. Dasso M. Modification in reverse: the SUMO proteases.Trends Biochem. Sci. 2007; 32: 286-295Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar]. Sumoylated species are low-abundant and short-lived, probably because of the high activity of SUMO peptidases in the cell [4Sacher M. Pfander B. Jentsch S. Identification of SUMO-protein conjugates.Methods Enzymol. 2005; 399: 392-404Crossref PubMed Scopus (19) Google Scholar]. Cohesin subunits are known to be modified by the small ubiquitin-like modifier (SUMO) [5Stead K. Aguilar C. Hartman T. Drexel M. Meluh P. Guacci V. Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion.J. Cell Biol. 2003; 163: 729-741Crossref PubMed Scopus (134) Google Scholar, 6Denison C. Rudner A.D. Gerber S.A. Bakalarski C.E. Moazed D. Gygi S.P. A proteomic strategy for gaining insights into protein sumoylation in yeast.Mol. Cell. Proteomics. 2005; 4: 246-254Crossref PubMed Scopus (222) Google Scholar, 7Potts P.R. Porteus M.H. Yu H.T. Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks.EMBO J. 2006; 25: 3377-3388Crossref PubMed Scopus (200) Google Scholar], but the physiological importance of this modification is currently unknown. Overexposure of western blots from extracts of Scc1-18myc tagged yeast cells reveals a few slow mobility forms of Scc1 that accumulate after DNA damage and are dependent on Ubc9 (Figure S1A available online). Pull-down experiments using a 6xhis-Flag (HF) N-terminal tag on SUMO confirmed that both the core and the more loosely associated subunits of the cohesin complex are sumoylated (Figure 1A ). Given that cohesin is subjected to a strict regulation by the cell cycle, we asked whether cohesin sumoylation also changes during cell-cycle progression. As shown in Figure 1B, Smc1 sumoylation peaks shortly after G1 release at 30°C, coincident with DNA replication (Figure 1B). Accordingly, Pds5 sumoylation has been shown to peak during DNA replication [5Stead K. Aguilar C. Hartman T. Drexel M. Meluh P. Guacci V. Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion.J. Cell Biol. 2003; 163: 729-741Crossref PubMed Scopus (134) Google Scholar], and Scc1 sumoylation is maximal during S phase, before its anaphase cleavage (Figure S1B), confirming that different cohesin subunits become SUMO targets during chromosome replication. We next examined the molecular requirements for cohesin sumoylation. Ubc9 can conjugate SUMO directly to the target protein, or in collaboration with E3 ligases. Sumoylation of Smc1 and Smc3 depend on the Mms21 ligase ([8Takahashi Y. Dulev S. Liu X. Hiller N.J. Zhao X. Strunnikov A. Cooperation of sumoylated chromosomal proteins in rDNA maintenance.PLoS Genet. 2008; 4: e1000215Crossref PubMed Scopus (57) Google Scholar] and data not shown). In contrast, Scc1 sumoylation is only marginally affected by the three known mitotic yeast E3 ligases (Figure 1C), indicating that individual cohesin subunits might differently depend on E3 ligases, at least under unperturbed conditions. An ATP binding and hydrolysis cycle by the nucleotide binding domain of the SMC subunits is necessary during cohesin loading and cohesion establishment [9Arumugam P. Gruber S. Tanaka K. Haering C.H. Mechtler K. Nasmyth K. ATP hydrolysis is required for cohesin's association with chromosomes.Curr. Biol. 2003; 13: 1941-1953Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 10Hu B. Itoh T. Mishra A. Katoh Y. Chan K.L. Upcher W. Godlee C. Roig M.B. Shirahige K. Nasmyth K. ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex.Curr. Biol. 2011; 21: 12-24Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]. The Smc1(K39I) mutant, which abrogates ATP binding (as well as interaction with Scc1), shows no detectable sumoylation (Figure 1D). An F584R mutation on the Smc1 protein that impairs SMC heterodimerization and loading onto chromatin (but not binding to Scc1) [11Mishra A. Hu B. Kurze A. Beckouët F. Farcas A.M. Dixon S.E. Katou Y. Khalid S. Shirahige K. Nasmyth K. Both interaction surfaces within cohesin's hinge domain are essential for its stable chromosomal association.Curr. Biol. 2010; 20: 279-289Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar] also shows no detectable sumoylation levels. In contrast, a mutant Smc1 protein that blocks ATP hydrolysis (E1158Q mutation) shows detectable, albeit diminished, levels of sumoylation (Figure 1D). The Smc1(E1158Q) protein can be transiently loaded onto chromatin, but does not entrap chromatin fibers and therefore cannot establish sister chromatid cohesion (SCC) [10Hu B. Itoh T. Mishra A. Katoh Y. Chan K.L. Upcher W. Godlee C. Roig M.B. Shirahige K. Nasmyth K. ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex.Curr. Biol. 2011; 21: 12-24Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]. Loading of cohesin onto chromosomes occurs during G1 and is mediated by a cohesin loader, the Scc2/4 complex [12Ciosk R. Shirayama M. Shevchenko A. Tanaka T. Toth A. Shevchenko A. Nasmyth K. Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins.Mol. Cell. 2000; 5: 243-254Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar]. As shown in Figure 1E, sumoylation of Scc1 also requires the activity of Scc2. Taken together, these results indicate that sumoylation of the cohesin complex takes place in a window between cohesin loading by Scc2/4 and chromatin entrapment during ATP hydrolysis. These results are in accordance with cohesin sumoylation occurring at the time of chromosome replication, and suggest that SUMO might play a role during the establishment of cohesion. Fusion of Ubc9 to a target protein has been recently shown to strongly induce sumoylation of the protein at its specific residue/s [13Jakobs A. Koehnke J. Himstedt F. Funk M. Korn B. Gaestel M. Niedenthal R. Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation.Nat. Methods. 2007; 4: 245-250Crossref PubMed Scopus (74) Google Scholar]. In order to assess a possible functional role for cohesin sumoylation, we decided to set up an analogous approach, consisting in fusion of the α-kleisin subunit of the cohesin complex to a SUMO deconjugating domain (Figure 2A ). We reasoned that this approach should result in downregulation of cohesin sumoylation levels without the need to simultaneously mutate all the modifiable cohesin subunits. Budding yeast cells code for two SUMO specific proteases, Ulp1 and Ulp2, and Ulp1 has been shown to have greater activity than Ulp2 in vitro [14Li S.J. Hochstrasser M. A new protease required for cell-cycle progression in yeast.Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar, 15Li S.J. Hochstrasser M. The yeast ULP2 (SMT4) gene encodes a novel protease specific for the ubiquitin-like Smt3 protein.Mol. Cell. Biol. 2000; 20: 2367-2377Crossref PubMed Scopus (311) Google Scholar]. Ulp1 is a 72 KDa protein, but only the last 200 amino acids of the protein code for a fully functional Ulp domain (UD). We therefore fused the C terminus of Scc1 to the UD of Ulp1. We engineered the fusions to be attached to a 3xHA tag, as a linker to allow the physical separation and proper folding of the two proteins. Overexpression of a Scc1-UD fusion lowered sumoylation of Smc1, Scc3 and Pds5 (Figure S2), indicating that the fusion can downregulate sumoylation of different subunits in the cohesin complex. In order to discard the possible global reduction in protein sumoylation due to overexpression of a SUMO peptidase, we placed the chimeras under the control of the SCC1 promoter. Additionally, the endogenous SCC1 gene was fused to an auxin induced degron (scc1-aid) [16Renshaw M.J. Ward J.J. Kanemaki M. Natsume K. Nédélec F.J. Tanaka T.U. Condensins promote chromosome recoiling during early anaphase to complete sister chromatid separation.Dev. Cell. 2010; 19: 232-244Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar], and the protein degraded before analyzing cohesin sumoylation. As shown in Figures 2B and 2C, the Scc1-UD fusion itself and the Smc3 subunit show reduced levels of sumoylation when expressed at physiological levels, indicating that downregulation of cohesin sumoylation is a local effect, and not the consequence of accumulation of a SUMO peptidase. Downregulation of cohesin ring sumoylation was dependent on the catalytic cysteine 580 in the Ulp domain, and its mutation to serine (Scc1-UDCS) allowed recovery of cohesin sumoylation levels (Figure 2C). In fact, this mutation not only restores but actually upregulates sumoylation of most cohesin subunits (Figures 2C and S2). The cohesin hypersumoylation detected in the Scc1-UDCS fusion could reflect the binding of the inactive domain to cohesin-SUMO conjugates, and the consequent block in deconjugation by the endogenous Ulp peptidases [17Li S.J. Hochstrasser M. The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity.J. Cell Biol. 2003; 160: 1069-1081Crossref PubMed Scopus (165) Google Scholar, 18Elmore Z.C. Donaher M. Matson B.C. Murphy H. Westerbeck J.W. Kerscher O. Sumo-dependent substrate targeting of the SUMO protease Ulp1.BMC Biol. 2011; 9: 74Crossref PubMed Scopus (26) Google Scholar]. In accordance with this hypothesis, sumoylation of Smc3 and Scc1-UD was restored to wild-type levels when the F474A mutation, known to prevent Ulp1 binding to SUMO [19Mossessova E. Lima C.D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.Mol. Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar], was introduced in the inactive UDCS domain (Scc1-UDFA,CS). These results indicate that fusion of the α-kleisin to a UD domain effectively alters the sumoylation state of assembled cohesin rings. In agreement with this, coimmunoprecipitation experiments confirmed that the Scc1-UD chimeras are able to interact with Scc3 (Figure 2D) and Smc3 (Figure 4E) with similar efficiencies as wild-type Scc1. These observations demonstrate that the Scc1-UD fusion does not affect the integrity of cohesin rings. Next, we analyzed the functionality of the UD fusion proteins. Expression of the SCC1-UD fusion from the GAL promoter is toxic in mcd1/ scc1 thermosensitive backgrounds (Figures S3A and S3C). The lethality of Scc1-UD overexpression is not simply due to increased nuclear levels of the Ulp1 domain, because a similarly expressed SMC5-UD fusion is not toxic but able to rescue growth of a thermosensitive SMC5 allele (Figure S3B; Smc5 is a subunit of cohesin-related SMC complex that is also modified by SUMO [20De Piccoli G. Torres-Rosell J. Aragón L. The unnamed complex: what do we know about Smc5-Smc6?.Chromosome Res. 2009; 17: 251-263Crossref PubMed Scopus (94) Google Scholar]). It is worth noting that the lethality is no longer observed when the Scc1-UD fusion is expressed at physiological levels from an SCC1 promoter (Figure 3A ); yet it does not complement the thermosensitive phenotype of scc1-73 cells, indicating that cohesin sumoylation is required for viability. In order to prove that these effects are due to the SUMO peptidase activity of the Ulp domain, we tested the growth of yeast cells expressing inactive versions of this moiety. As shown in Figure 3A, the Scc1-UD growth defects can be suppressed by inactivation of the SUMO peptidase domain (Scc1-UDCS and Scc1-UDFA,CS chimeras). Desumoylation is probably required to fine-tune the function of cohesin, because preventing binding of the inactive Ulp domain to SUMO (Scc1-UDFA,CS fusion) enables full rescue of the scc1-73 allele (Figure 3A) and normal levels of cohesin sumoylation (Figures 2B and 2C). Taken together, these results indicate that sumoylation, and to a lesser extent its deconjugation, are required for cell viability and cohesion function. One possible explanation for the Scc1-UD phenotypes is that downregulation of cohesin sumoylation might impair its binding to chromatin. In order to explore this possibility, we used chromatin fractionation to separate Triton X-100 soluble supernatant and chromatin pellet fractions. As shown in Figure 3B, we detected no difference in chromatin binding between the functional Scc1-UDFA,CS chimera and the nonsumoylated Scc1-UD fusion protein. Given that cohesin rings are properly assembled around an Scc1-UD fusion and efficiently bound to chromatin, but are not functional, we reasoned that downregulation of cohesin sumoylation might impair sister chromatid cohesion (SCC). scc1-73 cells were prearrested in G1 and released into a metaphase block after induction of the Scc1-UD chimeras. SCC was measured by evaluation of the levels of separated fluorescent chromosome tags inserted next to centromere 5. Expression of the SCC1-UD from the GAL promoter does not rescue the SCC defects of scc1-73 mutant cells (Figure 3C). This phenotype is dependent on UD binding to and deconjugating SUMO. Similar observations were made when the chimeras were expressed from the SCC1 promoter (Figure S3D). Sumoylation of cohesin takes place during S phase, after its loading onto chromatin and in a process that requires binding of ATP to the SMC heads. Therefore, our results strongly suggest that sumoylation of cohesin is required for its establishment, although we cannot formally exclude the possibility that SUMO is required for the maintenance of SCC. The establishment of SCC depends on acetylation of two lysine residues (K112 and K113) in Smc3 by Eco1/Ctf7 [21Ivanov D. Schleiffer A. Eisenhaber F. Mechtler K. Haering C.H. Nasmyth K. Eco1 is a novel acetyltransferase that can acetylate proteins involved in cohesion.Curr. Biol. 2002; 12: 323-328Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 22Skibbens R.V. Corson L.B. Koshland D. Hieter P. Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery.Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (384) Google Scholar]. Hence we tested the functional relation between acetylation and sumoylation of cohesin. First we performed SUMO pull-down experiments in eco1-1 thermosensitive cells. As shown in Figure 4A , Scc1 sumoylation levels were not affected by the presence of the eco1-1 mutation, neither at the permissive (23°C) nor after shift to the restrictive (37°C) temperatures. Sumoylation of the Smc3 subunit is also not affected by inactivation of Eco1 (Figure S4A), indicating that sumoylation is required before, or in parallel to, acetylation, for the establishment of SCC. Next, we tested the acetylation state of Smc3 in cells that are impaired in cohesin sumoylation. To this end, we first validated our anti-lysine antibodies, which recognize a band of the expected molecular weight in Smc3-3HA immunoprecipitates but not in the nonacetylated Smc3(K112R, K113R)-3HA double mutant (Figure 4B). We next checked Smc3 acetylation in cells that express the SCC1 gene fused to an auxin-inducible degron (scc1-aid). As expected, Smc3 acetylation levels lowered after degradation of the Scc1-aid protein during a 2 hr time course (Figure 4C). In contrast, expression of Scc1 or the Scc1-UD in scc1-aid cells allowed the maintenance of Smc3 acetylation levels, indicating that cohesin sumoylation is not required for its acetylation. Similar observations were made after inactivation of the ubc9-1 thermosensitive allele (Figure S4B). Although Smc3 acetylation levels do not depend on sumoylation of cohesin, we reasoned that the interaction between Scc1 and the acetylated form of Smc3 might become impaired when cohesin is not sumoylated. To discard the possible competition between the endogenous Scc1 protein and the UD chimeras for binding to acetylated Smc3, we first checked that Smc3 acetylation levels drop when the scc1-73 allele is inactivated by shift to the restrictive temperature (Figure 4D). We next introduced a second copy of the wild-type SCC1 or the SCC1-UD fusions in scc1-73 cells. As shown in Figure 4E, Scc1 and the Scc1-UD fusions are able to coimmunoprecipitate similar amounts of acetylated Smc3. Taken together, these results indicate that cohesin sumoylation is necessary neither for Smc3 acetylation nor for the interaction between Scc1 and acetylated Smc3. Therefore, both modifications, acetylation and sumoylation, must be required in parallel for the establishment of SCC. These results are surprising, because to our knowledge, this is the first case reported in which Smc3 is acetylated but has not yet established SCC. Cohesin acetylation is no longer required when the antiestablishment activity is eliminated. However, deletion of RAD61 in scc1-73 cells did not recover the growth defects of SCC1-UD expressing cells (Figure S4C), what indicates that sumoylation must promote SCC through a mechanism different from counteracting the antiestablishment activity. Here we have provided evidence that sumoylation of cohesin is required for the establishment of SCC. Although cohesin sumoylation occurs during S phase, it can also be triggered by DNA damage (Figure S1). Double-strand breaks stimulate cohesin loading and cohesion establishment out of S phase [23Ström L. Karlsson C. Lindroos H.B. Wedahl S. Katou Y. Shirahige K. Sjögren C. Postreplicative formation of cohesion is required for repair and induced by a single DNA break.Science. 2007; 317: 242-245Crossref PubMed Scopus (236) Google Scholar, 24Unal E. Heidinger-Pauli J.M. Koshland D. DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7).Science. 2007; 317: 245-248Crossref PubMed Scopus (252) Google Scholar], and this situation may similarly require the SUMO-dependent step for entrapment of sister chromatids (see [25McAleenan A. Cordon-Preciado V. Clemente-Blanco A. Liu I.-C. Sen N. Leonard J. Jarmuz A. Aragón L. SUMOylation of the α-kleisin subunit of cohesin is required for DNA damage-induced cohesion.Curr. Biol. 2012; (Published online July 5, 2012)https://doi.org/10.1016/j.cub.2012.06.045Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]). On the other hand, analysis of the Scc1-UD fusion, which is defective in cohesin sumoylation, shows that SUMO is essential for cohesion and cell viability. We currently cannot discard the possibility that Scc1-UD fusion might be affecting the sumoylation state of other nearby factors involved in SCC. We note that other reports have already pointed to a link between SUMO and cohesin [7Potts P.R. Porteus M.H. Yu H.T. Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks.EMBO J. 2006; 25: 3377-3388Crossref PubMed Scopus (200) Google Scholar, 8Takahashi Y. Dulev S. Liu X. Hiller N.J. Zhao X. Strunnikov A. Cooperation of sumoylated chromosomal proteins in rDNA maintenance.PLoS Genet. 2008; 4: e1000215Crossref PubMed Scopus (57) Google Scholar], and based on genetic evidence it has been proposed that Pds5 sumoylation would be detrimental for SCC [5Stead K. Aguilar C. Hartman T. Drexel M. Meluh P. Guacci V. Pds5p regulates the maintenance of sister chromatid cohesion and is sumoylated to promote the dissolution of cohesion.J. Cell Biol. 2003; 163: 729-741Crossref PubMed Scopus (134) Google Scholar]. Pds5 is a subunit that has apparent antagonistic roles in cohesin function [26Hartman T. Stead K. Koshland D. Guacci V. Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae.J. Cell Biol. 2000; 151: 613-626Crossref PubMed Scopus (225) Google Scholar, 27Panizza S. Tanaka T. Hochwagen A. Eisenhaber F. Nasmyth K. Pds5 cooperates with cohesin in maintaining sister chromatid cohesion.Curr. Biol. 2000; 10: 1557-1564Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 28Rowland B.D. Roig M.B. Nishino T. Kurze A. Uluocak P. Mishra A. Beckouët F. Underwood P. Metson J. Imre R. et al.Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity.Mol. Cell. 2009; 33: 763-774Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 29Sutani T. Kawaguchi T. Kanno R. Itoh T. Shirahige K. Budding yeast Wpl1(Rad61)-Pds5 complex counteracts sister chromatid cohesion-establishing reaction.Curr. Biol. 2009; 19: 492-497Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar]. This duality might be easily explained by a putative role of Pds5 in preventing ring opening [27Panizza S. Tanaka T. Hochwagen A. Eisenhaber F. Nasmyth K. Pds5 cooperates with cohesin in maintaining sister chromatid cohesion.Curr. Biol. 2000; 10: 1557-1564Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar], a process that would be inhibited during establishment, but shortly afterward reactivated to prevent DNA release. Similarly, sumoylation might promote cohesion through mechanisms that involve transient opening of the ring during the process of cohesion establishment at the replication fork. Since all subunits are conjugated to SUMO it is highly probable that sumoylation of different subunits will be redundant for establishment during a normal cell cycle. It has been proposed that Smc3 acetylation locks cohesin rings around sister chromatids [30Unal E. Heidinger-Pauli J.M. Kim W. Guacci V. Onn I. Gygi S.P. Koshland D.E. A molecular determinant for the establishment of sister chromatid cohesion.Science. 2008; 321: 566-569Crossref PubMed Scopus (329) Google Scholar, 31Rolef Ben-Shahar T. Heeger S. Lehane C. East P. Flynn H. Skehel M. Uhlmann F. Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion.Science. 2008; 321: 563-566Crossref PubMed Scopus (365) Google Scholar, 32Zhang J. Shi X. Li Y. Kim B.J. Jia J. Huang Z. Yang T. Fu X. Jung S.Y. Wang Y. et al.Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast.Mol. Cell. 2008; 31: 143-151Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar]. This modification persists until anaphase, to make closed cohesin rings refractory to the antiestablishment activity. Our results indicate that unsumoylated cohesin complexes are efficiently acetylated. Consequently, and based on current models, the Scc1-UD chimeras must be locked in the closed conformation; but, since they do not provide cohesion, unsumoylated rings might not embrace sister chromatids. Therefore, our results suggest that cohesin sumoylation is required transiently during chromosome replication to promote entrapment of the two sister chromatids. From this point of view, it is worth noting that the short-lived nature of the SUMO conjugates is perfectly suited for this purpose. A description of the methods and a list of strains used in this study (Table S1) can be found in the Supplemental Information. We thank all members in the Cell Cycle Lab for helpful discussion. Luis Aragon, Kim Nasmyth, Doug Koshland, Ethel Queralt, and Armelle Lengronne for reagents and strains. Work in J.T.-R. laboratory is supported by grants BFU2009-08808 and Consolider-Ingenio2010 from the Spanish Ministry of Science and Innovation. Download .pdf (.48 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S4, and Table S1" @default.
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• W2079405562 title "A SUMO-Dependent Step during Establishment of Sister Chromatid Cohesion" @default.
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